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Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique used to analyze the genome-wide methylation profiles on a single nucleotide level. This technique combines restriction enzymes and bisulfite sequencing in order to enrich for the areas of the genome that have a high CpG content. Due to the high cost and depth of sequencing needed to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 in order to reduce the amount of nucleotides needed to be sequenced to 1% of the genome.〔Alexander Meissner, Andreas Gnirke, George W. Bell, Bernard Ramsahoye, Eric S. Lander and Rudolf Jaenisch. 2005. "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis". ''Nucleic Acids Res''. 33(18):5868-77〕 The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches.〔Gu H, Smith ZD, Bock C, Boyle P, Gnirke A, Meissner A. 2011. "Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling." ''Nat Protoc''. 6(4):468-81. doi: 10.1038/nprot.2010.190.〕 ==Overview of protocol== #Enzyme digestion: First, genomic DNA is digested using a methylation-insensitive restriction enzyme. It is integral for the enzymes to not be influenced by the methylation status of the CpGs (sites within the genome where a cytosine is next to a guanine) as this allows for the digestion of both methylated and unmethylated areas. MspI is commonly used. This enzyme targets 3’CCGG5’ sequences and cleaves the phosphodiester bonds upstream of CpG dinucleotide. When using this particular enzyme, each fragment will have a CpG at each end. This digestion results in DNA fragments of various sizes. # End repair and A-tailing: Due to the nature of how MspI cleaves double stranded DNA, this reaction results in strands with sticky ends. End repair is necessary to fill in the 3’ terminal of the ends of the strands. The next step is adding an extra adenosine to both the plus and minus strands. This is referred to as A-Tailing and is necessary for adapter ligation in the subsequent step. End repair and A-Tailing is done within the same reactions, with dCTP, dGTP and dATP deoxyribonucleotides. In order to increase the efficiency of A tailing, the dATPs are added in excess in this reaction. # Sequence adapters: Methylated sequence adapters are ligated to the DNA fragments. The methylated adapter oligonucleotides have all cytosines replaced with 5’methyl-cytosines, in order to prevent the deamination of these cytosines in the bisulfite conversion reaction. For reactions to be sequenced using Illumina sequencers, the sequence adapters are used to hybridized to the adapters on the flow cell. # Fragment purification: The desired size of fragments is then selected to be purified. The different sizes of the fragments are separated using gel electrophoresis and are purified using gel excising. According to Gu et al., DNA fragments of 40-220 base pair are representative of the majority of promoter sequences and CpG islands〔 # Bisulfite conversion: The DNA fragments are then bisulfite converted, which is a process that deaminates unmethylated cytosine into a uracil. The methylated cytosines remain unchanged, due to the methyl group protecting them from the reaction. # PCR amplification: The bisulfite converted DNA is then amplified using PCR with primers that are complementary to the sequence adapters. # PCR purification: Before sequencing, the PCR product must be free of unused reaction reagents such as unincorporated dNTPs or salts. Thus, a step for PCR purification is required. This can be done by running another electrophoresis gel or by using kits designed specifically for PCR purification. # Sequencing: The fragments are then sequenced. When RRBS was first developed, Sanger sequencing was initially used. Now, next generation sequencing approaches are used. For Illumina sequencing, 36-base single-end sequencing reads are most commonly performed. # Sequence alignment and analysis: Due to the unique properties of RRBS, special software is needed for alignment and analysis.〔Chatterjee A, Rodger EJ, Stockwell PA, Weeks RJ, Morison IM. 2012. "Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries." ''J Biomed Biotechnol''. 2012:741542. doi: 10.1155/2012/741542〕 Using MspI to digest genomic DNA results in fragments that always start with a C (if the cytosine is methylated) or a T (if a cytosine was not methylated and was converted to a uracil in the bisulfite conversion reaction). This results in a non-random base pair composition. Additionally, the base composition is skewed due to the biased frequencies of C and T within the samples. Various software for alignment and analysis is available, such as Maq, BS Seeker, Bismark or BSMAP. Alignment to a reference genome allows the programs to identify base pairs within the genome that are methylated. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Reduced representation bisulfite sequencing」の詳細全文を読む スポンサード リンク
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